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Ralf Reski

Publications and source records attributed to Ralf Reski.

At least 19 recordsLinked to original sources
PubMed2006

The mechanism of gene targeting in Physcomitrella patens: homologous recombination, concatenation and multiple integration.

The model bryophyte Physcomitrella patens exhibits high frequencies of gene targeting when transformed with DNA constructs containing sequences homologous with genomic loci. 'Targeted gene replacement' (TGR) resulting from homologous recombination (HR) between each end of a targeting construct and the targeted locus occurs when either single or multiple targeting vectors are delivered. In the latter instance simultaneous, multiple, independent integration of different transgenes occurs at the targeted loci. In both single gene and 'batch' transformations, DNA can also be found to undergo 'targeted insertion' (TI), integrating at one end of the targeted locus by HR with one flanking sequence of the vector accompanied by an apparent non-homologous end-joining (NHEJ) event at the other. Untargeted integration at nonhomologous sites also occurs, but at a lower frequency. Molecular analysis of TI at a single locus shows that this occurs as a consequence of concatenation of the transforming DNA, in planta, prior to integration, followed by HR between a single site in the genomic target and two of its repeated homologues in the concatenated vector. This reinforces the view that HR is the major pathway by which transforming DNA is integrated in Physcomitrella.

Alleles↗
PubMed2006

Identification of genic moss SSR markers and a comparative analysis of twenty-four algal and plant gene indices reveal species-specific rather than group-specific characteristics of microsatellites.

BACKGROUND: The moss Physcomitrella patens is an emerging model in comparative plant science. At present, the Physcomitrella genome is sequenced at the Joint Genome Institute (USA). In this study we present our results on the development of expressed sequence tag-derived microsatellite markers for Physcomitrella patens, their classification and applicability as genetic markers on the intra- as well as on the interspecies level. We experienced severe restrictions to compare our results on Physcomitrella with earlier studies for other plant species due to varying microsatellite search criteria and a limited selection of analysed species. As a consequence, we performed a side by side analysis of expressed sequence tag-derived microsatellites among 24 plant species covering a broad phylogenetic range and present our results on the observed frequencies. RESULTS: We identified 3,723 microsatellites using the software MISA in a non-redundant Physcomitrella expressed sequence tag database comprising more than 37 megabases of nucleotide information. For 2,951 microsatellites appendant primer sequences have been derived. PCR of 376 microsatellites yielded 88 % successful amplicons and over 30 % polymorphisms between two Physcomitrella accessions. The polymorphism information content of 64 microsatellites based on 21 different Physcomitrella accessions was comparably high with a mean of 0.47 +/- 0.17. Of the 64 Physcomitrella microsatellite markers, 34 % respectively 79.7 % revealed cross-species applicability in two closely related moss species. In our survey of two green algae, two mosses, a fern, a fern palm, the ginkgo tree, two conifers, ten dicots and five monocots we detected an up to sevenfold variation in the overall frequency with a minimum of 37 up to maximal 258 microsatellites per megabase and a high variability among the different microsatellite class and motif frequencies. Numerous species-specific microsatellite frequencies became evident and several deviations to earlier reports were ascertained. CONCLUSION: With the Physcomitrella microsatellite marker set a valuable tool has been made available for further genetic and genomic applications on the intra- as well as on the interspecies level. The comparative survey of expressed sequence tag-derived microsatellites among the plant kingdom is well suited for a classification of future studies on plant microsatellites.

Base Pairing↗
PubMed2006

Biosynthesis of C9-aldehydes in the moss Physcomitrella patens.

After wounding, the moss Physcomitrella patens emits fatty acid derived volatiles like octenal, octenols and (2E)-nonenal. Flowering plants produce nonenal from C18-fatty acids via lipoxygenase and hydroperoxide lyase reactions, but the moss exploits the C20 precursor arachidonic acid for the formation of these oxylipins. We describe the isolation of the first cDNA (PpHPL) encoding a hydroperoxide lyase from a lower eukaryotic organism. The physiological pathway allocation and characterization of a downstream enal-isomerase gives a new picture for the formation of fatty acid derived volatiles from lower plants. Expression of a fusion protein with a yellow fluorescent protein in moss protoplasts showed that PpHPL was found in clusters in membranes of plastids. PpHPL can be classified as an unspecific hydroperoxide lyase having a substrate preference for 9-hydroperoxides of C18-fatty acids but also the predominant substrate 12-hydroperoxy arachidonic acid is accepted. Feeding experiments using arachidonic acid show an increase in the 12-hydroperoxide being metabolized to C8-aldehydes/alcohols and (3Z)-nonenal, which is rapidly isomerized to (2E)-nonenal. PpHPL knock out lines failed to emit (2E)-nonenal while formation of C8-volatiles was not affected indicating that in contrast to flowering plants, PpHPL is only involved in formation of a specific subset of volatiles.

Aldehyde-Lyases↗
PubMed2006

Rapid alteration of the phosphoproteome in the moss Physcomitrella patens after cytokinin treatment.

Cytokinin hormones are crucial regulators of a large number of processes in plant development. Recently, significant progress has been made toward the elucidation of the molecular details of cytokinin that has led to a model for signal transduction involving a phosphorylation cascade. However, the current knowledge of cytokinin action remains largely unknown and does not explain the different roles of this hormone. To gain further insights into this aspect of cytokinin action and the inducible phosphorelay, we have produced the first large-scale map of a phosphoproteome in the moss Physcomitrella patens. Using a protocol that we recently published (Heintz, D.; et al. Electrophoresis 2004, 25, 1149-1159) that combines IMAC, MALDI-TOF-MS, and LC-MS/MS, a total of 172 phosphopeptide sequences were obtained by a peptide de novo sequencing strategy. Specific P. patens EST and raw genomic databases were interrogated, and protein homology searches resulted in the identification of 112 proteins that were then classified into functional categories. In addition, the temporal dynamics of the phosphoproteome in response to cytokinin stimulation was studied at 2, 4, 6, and 15 min after hormone addition. We identified 13 proteins that were not previously known targets of cytokinin action. Among the responsive proteins, some were involved in metabolism, and several proteins of unknown function were also identified. We have mapped the time course of their activation in response to cytokinin and discussed their hypothetical biological significance. Deciphering these early induced phosphorylation events has shown that the cytokinin effect can be rapid (few minutes), and the duration of this effect can be variable. Also phosphorylation events can be differentially regulated. Taken together our proteomic study provides an enriched look of the multistep phosphorelay system mediating cytokinin response and suggests the existence of a multidirectional interaction between cytokinin and numerous other pathways.

Bryopsida↗
PubMed2005

Use of endogenous signal sequences for transient production and efficient secretion by moss (Physcomitrella patens) cells.

BACKGROUND: Efficient targeting to appropriate cell organelles is one of the bottlenecks for the production of recombinant proteins in plant systems. A common practice is to use the native secretory signal peptide of the heterologous protein to be produced. Though general features of secretion signals are conserved between plants and animals, the broad sequence variability among signal peptides suggests differing efficiency of signal peptide recognition. RESULTS: Aiming to improve secretion in moss bioreactors, we quantitatively compared the efficiency of two human signal peptides and six signals from recently isolated moss (Physcomitrella patens) proteins. We therefore used fusions of the different signals to heterologous reporter sequences for transient transfection of moss cells and measured the extra- and intracellular accumulation of the recombinant proteins rhVEGF and GST, respectively. Our data demonstrates an up to fivefold higher secretion efficiency with endogenous moss signals compared to the two utilised human signal peptides. CONCLUSION: From the distribution of extra- and intracellular recombinant proteins, we suggest translational inhibition during the signal recognition particle-cycle (SRP-cycle) as the most probable of several possible explanations for the decreased extracellular accumulation with the human signals. In this work, we report on the supremacy of moss secretion signals over the utilised heterologous ones within the moss-bioreactor system. Though the molecular details of this effect remain to be elucidated, our results will contribute to the improvement of molecular farming systems.

Amino Acid Sequence↗
PubMed2005

A fast and flexible PEG-mediated transient expression system in plants for high level expression of secreted recombinant proteins.

Plant expression systems offer a valuable alternative to traditional systems for the production of recombinant biopharmaceuticals. A highly efficient polyethyleneglycol (PEG)-mediated transient expression system for secreted recombinant proteins in plants has been developed. The human vascular endothelial growth factor 121 (rhVEGF) has been successfully expressed and efficiently secreted into the culture medium by transiently transformed moss protoplasts. In order to obtain secretion efficiency data, different expressed signal peptides were analysed and time course studies were performed with expression constructs containing different promoters. The transformation procedure was optimised for high level expression (up to 10 microg/ml) and successfully performed even with a transgenic glyco-engineered strain lacking plant-specific immunogenic sugar residues in N-glycans. The amount of rhVEGF was produced in such quantity that it allowed for the analysis of biological activity, silver-staining and Western blotting, revealing the correct formation and processing of the homodimer. This fast and flexible transient expression system enables feasibility studies and construct optimisation to be concluded within a few days, thus avoiding the time consuming step of having to generate stably transformed lines.

Bryopsida↗
PubMed2005

Protein encoding genes in an ancient plant: analysis of codon usage, retained genes and splice sites in a moss, Physcomitrella patens.

BACKGROUND: The moss Physcomitrella patens is an emerging plant model system due to its high rate of homologous recombination, haploidy, simple body plan, physiological properties as well as phylogenetic position. Available EST data was clustered and assembled, and provided the basis for a genome-wide analysis of protein encoding genes. RESULTS: We have clustered and assembled Physcomitrella patens EST and CDS data in order to represent the transcriptome of this non-seed plant. Clustering of the publicly available data and subsequent prediction resulted in a total of 19,081 non-redundant ORF. Of these putative transcripts, approximately 30% have a homolog in both rice and Arabidopsis transcriptome. More than 130 transcripts are not present in seed plants but can be found in other kingdoms. These potential "retained genes" might have been lost during seed plant evolution. Functional annotation of these genes reveals unequal distribution among taxonomic groups and intriguing putative functions such as cytotoxicity and nucleic acid repair. Whereas introns in the moss are larger on average than in the seed plant Arabidopsis thaliana, position and amount of introns are approximately the same. Contrary to Arabidopsis, where CDS contain on average 44% G/C, in Physcomitrella the average G/C content is 50%. Interestingly, moss orthologs of Arabidopsis genes show a significant drift of codon fraction usage, towards the seed plant. While averaged codon bias is the same in Physcomitrella and Arabidopsis, the distribution pattern is different, with 15% of moss genes being unbiased. Species-specific, sensitive and selective splice site prediction for Physcomitrella has been developed using a dataset of 368 donor and acceptor sites, utilizing a support vector machine. The prediction accuracy is better than those achieved with tools trained on Arabidopsis data. CONCLUSION: Analysis of the moss transcriptome displays differences in gene structure, codon and splice site usage in comparison with the seed plant Arabidopsis. Putative retained genes exhibit possible functions that might explain the peculiar physiological properties of mosses. Both the transcriptome representation (including a BLAST and retrieval service) and splice site prediction have been made available on http://www.cosmoss.org, setting the basis for assembly and annotation of the Physcomitrella genome, of which draft shotgun sequences will become available in 2005.

Alternative Splicing↗
PubMed2005

Cloning and expression of the tobacco CHLM sequence encoding Mg protoporphyrin IX methyltransferase and its interaction with Mg chelatase.

S-adenosyl-L-methionine:Mg-protoporphyrin IX methyltransferase (MgPMT) is an enzyme in the Mg branch of the tetrapyrrole biosynthetic pathway. The nucleotide sequence of tobacco (Nicotiana tabacum) CHLM was identified and the cDNA sequence was used to express the precursor, the mature and a truncated recombinant MgPMT for enzymatic activity tests and for the formation of polyclonal antibodies. Comparison of the mature and the truncated MgPMT revealed three critical amino acids at the N-terminus of MgPMT for the maintenance of enzyme activity. To assess the contribution of CHLM expression to the control of the metabolic flow in the tetrapyrrole pathway, CHLM transcripts and protein levels, the enzyme activity and the steady-state levels of Mg protoporphyrin and Mg protoporphyrin monomethylester were analysed during greening of seedlings and plant development as well as under day/night and continuous growth conditions. These expression studies revealed posttranslational activation of MgPMT during greening and light/dark-cycles. Using the yeast two-hybrid system physical interaction was demonstrated between MgPMT and the CHLH subunit of Mg chelatase. Activity of recombinant MgPMT expressed in yeast cells was stimulated in the presence of the recombinant CHLH subunit. Implications for posttranslational regulation of MgPMT are discussed for the enzymatic steps at the beginning of the Mg branch.

Amino Acid Sequence↗
PubMed2005

Moss (Physcomitrella patens) functional genomics--Gene discovery and tool development, with implications for crop plants and human health.

Recently, the moss Physcomitrella patens was established as a versatile tool in plant functional genomics. Mosses represent the oldest living clade of land plants, separated by approximately 450 million years of evolution from crop plants. Consequently, mosses contain metabolites and genes not known from these seed plants. In Physcomitrella, nuclear genes can be targeted by homologous recombination as efficiently as in yeast, allowing reverse genetics approaches in plants at high-throughput levels for the first time. Comprehensive expressed sequence tag databases gave new insights into the levels of diversity in land plants which are now ready to be exploited in plant biotechnology. In forward genetics screens, saturated tagged mutant collections help to unravel novel gene - function relationships. Additionally, proteomics tools are at hand to analyse subcellular proteomes, as well as the phosphoproteome, as the core of eukaryotic signal transduction. Moreover, specifically designed Physcomitrella strains can produce human therapeutic proteins safely and cost-effectively in bioreactors.

Bioreactors↗
PubMed2005

Gene clusters involved in anaerobic benzoate degradation of Geobacter metallireducens.

The degradation of aromatic compounds follows different biochemical principles in aerobic and anaerobic microorganisms. While aerobes dearomatize and cleave the aromatic ring by oxygenases, facultative anaerobes utilize an ATP-dependent ring reductase for the dearomatization of the activated key intermediate benzoyl-coenzyme A (CoA). In this work, the aromatic metabolism was studied in the obligately anaerobic model organism Geobacter metallireducens. The gene coding for a putative carboxylic acid-CoA ligase was heterologously overexpressed and the gene product was characterized as a highly specific benzoate-CoA ligase catalysing the initial step of benzoate metabolism. However, no evidence for the presence of an ATP-dependent benzoyl-CoA reductase as observed in facultative anaerobes was obtained. In a proteomic approach benzoate-induced proteins were identified; the corresponding genes are organized in two clusters comprising 44 genes. Induction of representative genes during growth on benzoate was confirmed by reverse transcription polymerase chain reaction. The results obtained suggest that benzoate is activated to benzoyl-CoA, which is then reductively dearomatized to cyclohexa-1,5-diene-1-carbonyl-CoA, followed by beta-oxidation reactions to acetyl-CoA units, as in facultatively anaerobic bacteria. However, in G. metallireducens the process of reductive benzene ring dearomatization appears to be catalysed by a set of completely different protein components comprising putative molybdenum and selenocysteine containing enzymes.

Acyl Coenzyme A↗
PubMed2005

Enhanced recovery of a secreted recombinant human growth factor using stabilizing additives and by co-expression of human serum albumin in the moss Physcomitrella patens.

The production of pharmaceutical proteins in plants provides a valuable alternative to other traditional eukaryotic expression systems from economic and safety perspectives. The moss Physcomitrella patens allows the expression and secretion of complex target proteins into a simple aqueous maintenance medium, which facilitates downstream processing by rendering it less complex. To address the question of whether the addition of protein-stabilizing substances enhances the recovery of a target protein secreted into the culture medium, several additives at different concentrations were tested in a small-scale screening system. Although polyvinylpyrrolidone (PVP) and human serum albumin (HSA) showed a significant impact on protein levels, supplementation of the medium with these substances was accompanied by certain limitations in upstream processes, such as foam formation (HSA), and in downstream processes, such as reduced binding efficiency on chromatography columns (PVP), respectively. In order to reap the benefit of the enhancing effect and to avoid the given negative aspects, we developed a new strategy based on the recombinant expression of HSA in plants that are already capable of expressing a target protein. First, we analysed the expression and secretion of recombinant HSA in transiently and stably transformed wild-type (WT) plants. HSA was then co-expressed in Physcomitrella plants transgenic for human vascular endothelial growth factor (VEGF). Even with high expression levels of recombinant human VEGF (rhVEGF), the co-expression of recombinant HSA (rHSA) resulted in 48%-102% higher recovery of the target protein without concomitant negative effects on the upstream process. This strategy enables the enhanced recovery of target protein and does not require the addition of foreign components directly to the culture medium.

Journal Article↗
PubMed2004

Isolation and characterisation of three moss-derived beta-tubulin promoters suitable for recombinant expression.

The moss Physcomitrella patens is an excellent tool to study plant gene-function relationships due to its high rate of homologous recombination (HR). It has also been shown to be very useful in the production of recombinant proteins which are secreted into a simple medium. Thus, there is a need for suitable promoters functional in this well established model organism. We isolated genomic flanking regions of the beta-tubulin gene family from Physcomitrella, concentrating on those family members showing high transcript abundance integrated over gametophytic tissues. Using a novel, fast and reliable quantification assay based on the transient expression and secretion of a recombinant human protein, three genomic upstream regions were characterised in serial deletion constructs. Expression rates were up to three times higher than those obtained with the 35S cauliflower mosaic virus (35S) promoter, which served as a reference.

5' Untranslated Regions↗
PubMed2004

From axenic spore germination to molecular farming. One century of bryophyte in vitro culture.

The first bryophyte tissue culture techniques were established almost a century ago. All of the techniques that have been developed for tissue culture of seed plants have also been adapted for bryophytes, and these range from mere axenic culture to molecular farming. However, specific characteristics of bryophyte biology--for example, a unique regeneration capacity--have also resulted in the development of methodologies and techniques different than those used for seed plants. In this review we provide an overview of the application of in vitro techniques to bryophytes, emphasising the differences as well as the similarities between bryophytes and seed plants. These are discussed within the framework of physiological and developmental processes as well as with respect to potential applications in plant biotechnology.

Bryophyta↗
PubMed2004

A large plant beta-tubulin family with minimal C-terminal variation but differences in expression.

Tubulins, as the major structural component of microtubules (MT), are highly conserved throughout the entire eukaryotic kingdom. They consist of alpha/beta heterodimers. Both monomers, at least in multicellular organisms, are encoded by gene families. In higher plants up to eight beta-tubulin isotypes, mostly differing in their very C-termini, have been described. These variable beta-tubulin C-termini have been discussed in the context of functional microtubule diversity. However, in plants, in contrast to vertebrates, functional isotype specificity remains yet to be demonstrated. Unlike higher plants, unicellular green algae in general do not exhibit isotypic variations. The moss Physcomitrella patens is a phylogenetic intermediate between higher plants and green algae. We isolated six beta-tubulin genes from Physcomitrella, named PpTub1 to 6. We show that the exon/intron structure, with the exception of one additional intron in PpTub6, is identical with that of higher plants, and that some members of the family are differentially expressed. Moreover, we find that all Physcomitrella isotypes are highly conserved and, most strikingly, are almost identical within their C-terminal amino acids (aa). This evolutionary ancient and large beta-tubulin gene family without significant isotypic sequence variation points to a role of differential regulation in the evolution of plant tubulin isotypes.

Amino Acid Sequence↗
PubMed2004

Physcomitrella patens is highly tolerant against drought, salt and osmotic stress.

In order to determine the degree of tolerance of the moss Physcomitrella patens to different abiotic stress conditions, we examined its tolerance against salt, osmotic and dehydration stress. Compared to other plants like Arabidopsis thaliana, P. patens exhibits a high degree of abiotic stress tolerance, making it a valuable source for the identification of genes effecting the stress adaptation. Plants that had been treated with NaCl tolerated concentrations up to 350 mM. Treatments with sorbitol revealed that plants are able to survive concentrations up to 500 mM. Furthermore, plants that had lost 92% water on a fresh-weight basis were able to recover successfully. For molecular analyses, a P. patens expressed sequence tag (EST) database was searched for cDNA sequences showing homology to stress-associated genes of seed plants and bacteria. 45 novel P. patens genes were identified and subjected to cDNA macroarray analyses to define their expression pattern in response to water deficit. Among the selected cDNAs, we were able to identify a set of genes that is specifically up-regulated upon dehydration. These genes encode proteins exerting their function in maintaining the integrity of the plant cell as well as proteins that are known to be members of signaling networks. The identified genes will serve as molecular markers and potential targets for future functional analyses.

Bryopsida↗
PubMed2004

Quantitative promoter analysis in Physcomitrella patens: a set of plant vectors activating gene expression within three orders of magnitude.

BACKGROUND: In addition to studies of plant gene function and developmental analyses, plant biotechnological use is largely dependent upon transgenic technologies. The moss Physcomitrella patens has become an exciting model system for studying plant molecular processes due to an exceptionally high rate of nuclear gene targeting by homologous recombination compared with other plants. However, its use in transgenic approaches requires expression vectors that incorporate sufficiently strong promoters. To satisfy this requirement, a set of plant expression vectors was constructed and equipped with either heterologous or endogenous promoters. RESULTS: Promoter activity was quantified using the dual-luciferase reporter assay system. The eight different heterologous promoter constructs tested exhibited expression levels spanning three orders of magnitude. Of these, the complete rice actin1 gene promoter showed the highest activity in Physcomitrella, followed by a truncated version of this promoter and three different versions of the cauliflower mosaic virus 35S promoter. In contrast, the Agrobacterium tumefaciens nopaline synthase promoter induced transcription rather weakly. Constructs including promoters commonly used in mammalian expression systems also proved to be functional in Physcomitrella. In addition, the 5' -regions of two Physcomitrella glycosyltransferases (i.e. alpha1,3-fucosyltransferase and beta1,2-xylosyltransferase) were identified and functionally characterised in comparison to the heterologous promoters. Furthermore, motifs responsible for enhancement of translation efficiency - such as the TMV omega element and a modified sequence directly prior the start codon - were tested in this model. CONCLUSION: We developed a vector set that enables gene expression studies, both in lower and higher land plants, thus providing valuable tools applicable in both basic and applied molecular research.

Bryopsida↗
PubMed2004

An efficient protocol for the identification of protein phosphorylation in a seedless plant, sensitive enough to detect members of signalling cascades.

We describe a reproducible protocol to explore for the first time the phosphoproteome of a seedless plant, the moss Physcomitrella patens. Following tryptic digestion of a total protein extract, phosphorylated peptides were isolated using the combination of C18 reverse-phase chromatography (RP-C18), immobilized Fe(3+) metal affinity chromatography (IMAC), capillary zone electrophoresis (CZE), liquid chromatography-tandem mass spectrometry (LC-MS/MS) and matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) analysis. The total protein extracts were first prepared as usually made for plant two-dimensional gel electrophoresis, the tryptic digest was desalted and concentrated by reverse phase chromatography, and from this mixture the phosphorylated peptides were captured by IMAC. Subsequently, the complex phosphopeptide mixture was separated into ten fractions by RP-C18-HPLC and each analyzed by CZE. This permitted the detection of 253 distinct phosphopeptides. These were identified by nano-LC-MS/MS and MALDI-TOF-MS analysis in conjunction with alkaline phosphatase treatment to remove covalently bound phosphate to specifically identify the phosphopeptides. Among others, several kinases and a transcription factor were identified. This protocol will be taken as a basis to unravel early events in plant signal transduction known to occur via rapid phosphorylation/dephosphorylation of proteins.

Bryopsida↗